Bonding tissues and cross-linking proteins with naphthalimide compounds

ABSTRACT

Naphthalimide compounds are used in tissue bonding and protein cross-linking applications. When activated by an activating agent, such as light in the 400-500 nm absorption range, the naphthalimide compounds form chemically-reactive species that cross-link proteins, bond connective tissues together, and bond tissues and other biomaterials together. A naphthalimide-labeled biomolecule, such as a naphthalimide-labeled chitosan, is also capable of bonding tissues without subsequent direct illumination of the contacted tissue area. The naphthalimide compounds may be used in tissue or arterial repair, stabilization of an expanded arterial wall after angioplasty, tethering pharmaceutical agents to tissue surfaces to provide local drug delivery, and for chemically bonding skin care products, sunscreens, and cosmetics to the skin.

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 60/517,618, entitled “BONDING TISSUES AND CROSS-LINKINGPROTEINS WITH NAPHTHALIMIDE COMPOUNDS” filed on Nov. 5, 2003, havingRonald E. Utecht, Kaia L. Kloster, Millard M. Judy, Kevin J. Vaska, andJames L. Matthews, listed as the inventor(s), the entire content ofwhich is hereby incorporated by reference.

BACKGROUND

This invention relates to naphthalimide compounds and their use intissue bonding and protein cross-linking. This invention also pertainsto devices and methods for arterial repair, preservation of expandedinternal luminal diameters, and local delivery of drugs, skin carematerials, sunscreens, and cosmetics.

Wound closure in body tissues while maintaining low levels ofinflammation with resulting granuloma formation and attaining patencyagainst leakage across the walls of luminal structures such as bloodvessels remains a significant problem in surgical and trauma practice.Current closure practices involving sutures or mechanical devices suchas clips, staples, or nails result in the introduction of foreignmaterials, which are sources of foreign body reactions and inflammation,and the formation of holes through luminal walls by the closing agent,which serve as potential avenues of post-operative fluid leakage andloss of luminal patency.

From catgut to synthetic polymers, sutures have been the traditionaltool for vascular repair. However, fistulas and granulomas can form as aresult of intolerance to the suture material. Suture techniques can alsoresult in smaller residual lumens and reduced perfusion. These sideeffects can lead to necrosis, healing disorders, and ultimate dehiscenceof the wound. Furthermore, leakage from the needle puncture sites can beproblematic, particularly in cerebral applications or in patients with acompromised ability to achieve hemostasis (i.e. hemophiliacs or patientsundergoing anticoagulant therapy). Finally, suture techniques aretedious and time-consuming, requiring a concerted effort on the part ofthe surgeon and therefore contributing to overall expense.

Mechanical assists such as staples and vascular clips have been proposedto facilitate tissue repair. While they do shorten operative times, theassociated expense and potential risk of clip failure raise questionsregarding their benefits over sutures. Furthermore, some staples requireremoval and may be associated with more patient discomfort.

Laser thermal tissue welding experiments report mixed results inachieving tissue bonds. Numerous infrared wavelengths, including thoseof the Nd:YAG, Argon, and CO₂ lasers, have been tried. Laser welding hasproven to be an exacting methodology, where insufficient exposuresresult in ineffective tissue bonding and high temperatures areassociated with tissue destruction. In fact, the requisite denaturationof proteins (with tissue temperatures in the range of 60-80° C.) andassociated collateral thermal damage appear to be the primary limitingfactors for this technique.

Inflammation arising from foreign-material based wound closing agentscan result, for example, in sufficient scarring to seriously impedefunction such as by imposing a barrier to laminar blood flow in a bloodvessel possibly leading to clot formation and subsequent complications,or by degrading the desired cosmetic effects in skin plastic or traumarepair surgery.

Tissue adhesives comprising protein species, synthetic polymers, andbiological materials have been advocated for wound repair to eliminateor minimize mechanical or foreign body effects. Protein based systemssuch as fibrin solutions and sprays offer hemostasis but little in theway of mechanical strength in holding opposing surfaces together.Synthetic polymeric glues such as polylactates and polyglycolates offermechanical strength, but their products of chemical attachment in tissueare toxic and inflammatory. Acrylic based cements offer strength but areconfined to external use on skin wounds because they are toxic and as afilm impede migration of molecular and cellular species across bondedsurfaces. Tissue adhesives incorporating aldehyde based proteincross-linking agents such as BioGlue™ have been used. However, long termdiffusion of the aldehyde species away from the binding site leads todeleterious inflammation and granuloma formation.

The concept of a “patch” is also known. Various vascular repairprocedures, notably carotid endarterectomy closure, have utilizednumerous patch materials. It is important to note that this type ofpatching requires tailored fitting and extensive suturing to repair thesite of injury. However, there are some associated benefits. The use ofa patch helps avoid residual stenosis and decreases the likelihood ofrestenosis. Furthermore, a patch makes for easier closure under thesedifficult conditions and suffers less perioperative thrombosis. The sizeand shape of the patch are important to long-term success. A patch thatis too large can lead to increases in wall stress and ultimate dilationor rupture. Large deviations from the native lumen size can also lead toincreases in turbulence in blood flow, often associated with low shearrates and progression of the atherosclerotic process in arteries thatare so predisposed. Experience would suggest that a long, tapered,panhandle shaped patch serves better than an oval patch to maximize thebenefits and avoid potential risks.

What is needed is a method of applying a patch over an arterial lesionwhich achieves structural competency and hemostasis without attendantleakage of blood through the luminal wall and patch, granulomatoustissue growth into the vessel lumen, decrease in luminal area due toforeign body reaction, and initiation of intraluminal clot formation.

Prior tissue bonding technology using 4-amino-1,8-naphthalimidebiomolecular cross-linking has successfully achieved tissue closurewithout inflammatory reactions or penetration by foreign objects. (U.S.Pat. Nos. 5,235,045; 5,565,551; 5,766,600; 5,917,045; and 6,410,505; thecontent of each of these patents is incorporated by reference herein).This tissue bonding technology requires the application of light havinga wavelength within the absorption spectrum of 400-500 nm (blue light)to the photochemical upon the tissue or biomaterial surfaces in order toinitiate the photochemical bonding process. Minimization of lightrequirements would facilitate the ease of use for clinicians.

What is also needed, therefore, is a means of attaching two tissuesurfaces together or a tissue surface to a compatible biomaterial toeffect wound closure that does not introduce a material that induces aninflammatory reaction or compromise the structural integrity of aluminal wall. What is further needed is a means of attaching two tissuesurfaces or a tissue surface and a compatible biomaterial that does notrequire direct application of light to the tissue surfaces beingattached.

Concerns also exist for the long-term retention of the opened arteriallumen after balloon dilation during percutaneous transluminal coronaryangioplasty (“PTCA”), which is limited by processes that lead tore-occlusion within 3-6 months. PTCA has been one of the primarytreatment modalities for revascularization of arterial stenoses.However, two aspects of PTCA have motivated cardiologists to seekalternative methods of treating the coronary stenosis: (1) acuteischemic complications related to vessel injury and the PTCA procedureitself, and (2) the occurrence of late restenosis, or reclosure of thetreated site.

The occurrence of restenosis, or reclosure of the dilated vessel within3-6 months of treatment, is the primary problem arising from the PTCAtreatment and appears to be related to vascular injury. Damage to thevessel wall can lead to the release of thrombogenic, chemotactic, andgrowth factors. Endothelial denudation promotes platelet aggregation,thrombus formation, and activation of macrophages, lymphocytes, andsmooth muscle cells. Activated platelets proceed to release additionalmitogens including platelet derived growth factor (“PDGF”), fibroblastgrowth factor (“FGF”), and epidermal growth factor (“EGF”). Anothercontributing factor to loss of luminal diameter is the passive processof elastic recoil. The elastic nature of the vasculature promotes returnto its original dimensions and can account for a significant loss ofinitial diameter gain. The excessive reparative response, compounded byelastic recoil, can become occlusive in itself propagating symptomaticrecurrence including myocardial ischemia and angina. Alterations inlocal rheology such as turbulence and elevated shear stresses have alsobeen associated with the restenosis process.

A significant decrease in numbers and rates of re-occlusion has beenobtained by use of a mechanical cylindrically-shaped device, a stent,which maintains the expanded lumen against recoil and remodeling.Stents, which are typically made of a biocompatible metal, becomeincorporated within the vascular wall upon re-growth of the endotheliumand are not removable. This feature can compromise re-treatment ortreatment of distal portions of the stented vessel. Metallic stents caninitiate a thrombogenic and immunogenic response, such as a foreign bodyresponse with inflammation. Moreover, metal stents have limitedflexibility, making them difficult to deploy in smaller vessels. Becausemetal stents are permanent, their continued presence may interfere withfuture interventions and may lead to corrosion, perforation, andpotential aneurysm. On an individual basis, the various metals beingused may cause an allergic reaction.

Second generation stents have been developed in an attempt to addressthe problems listed above. Temporary metallic stents address the issueof permanence, but excessive trauma is associated with the retrievalprocess. Stent coatings, such as genetically engineered endothelialcells or various polymers have been employed in an attempt to reducethrombogenicity. Polymers such as nylon, silicone, polyurethane, andfibrin have been tested with mixed results. Though data suggest somereduction in thrombus formation, other problems, including donorinfection, optimization of formulation and delivery, and immunologicalresponse remain to be addressed. Stents comprised entirely of polmericmaterial offer an alternative to metallic stents. However, deploymenttechniques requiring heat, such as that required for polycaprolactone,can cause denaturation of adjacent tissues, and acidic breakdownproducts of biodegradable polymers can cause a significant inflammatoryresponse. An additional consideration with biodegradable stents is thepotential for atrophy of the musculoelastic elements in the arterialwall while the stent is in place, which may lead to aneurismaldilatation after the stent has been degraded. Finally, the polymerstents are intrinsically weaker than their metallic counterparts andadditional bulk may be required to achieve adequate hoop strength.

Drugs capable of inhibiting thrombus formation and/or neointimalproliferation can be utilized, but systemic delivery of severalappropriate and promising pharmaceutical agents has failed to demonstateclinical significance in reducing restenosis. This could result from afailure to achieve adequate local doses because of the toxic effect ofhigh systemic delivery. Local delivery results in high localconcentrations (up to ten times systemic concentrations) while avoidingtoxicity. Polymeric stents or stent coatings can be used to incorporateor bind drugs with ensuing controlled, sustained, local drug delivery atthe site of vascular injury.

Pharmaceutical coated stents are presently in the market and are beingincreasingly used. By attaching antithrombotic or antiproliferativepharmaceutical agents to the stent surface, reductions in restentosisrates have been reported. However, the mode of drug attachment can alterthe biological activity of the compound, possibly due to masking ofactive sites or undesirable conformational changes. Furthermore, stentsgenerally cover less than 10% of targeted vessel wall segments,resulting in nonuniform delivery to the arterial wall. Recent reportssuggest an unfavorably high rate of allergic reactions and occlusivethrombotic responses to the coated stents.

What is needed, therefore, is a method for stabilizing the dilatedvascular wall without the introduction of a foreign body, and also formaintaining the diameter of an artery expanded through balloon dilationin order to restore and maintain blood flow. What is also needed is amethod for providing targeted, local drug delivery to the site ofarterial expansion. Ideally, such a method should minimize the risks ofrestenosis and immune response. Such a method would also be useful forthe local delivery of drugs, skin care materials, sunscreens, andcosmetics to the skin and to other anatomical, physical, surgical, andmedical sites.

SUMMARY

This invention is directed to naphthalimide compounds and their use intissue bonding and protein cross-linking. This invention also pertainsto devices and methods for tissue and arterial repair, preservation ofexpanded internal luminal diameters, and local delivery of drugs, skincare materials, sunscreens, and cosmetics. In particular, the presentinvention utilizes naphthalimide compounds, which produce an adhesiveagent when applied to the surface of a biomaterial and activated by anactivating agent. The present invention also particularly relates tonaphthalimide labeled biomolecules that may be used to link tissuesurfaces together without direct activation of the contacted tissue areawith an activating agent, such as light energy.

One aspect of this invention particularly pertains to naphthalimidecompounds. Upon activation by an activating agent in an environmentindependent of the presence or absence of oxygen, naphthalimidecompounds generate activated species. The activated species can causestructural changes in lipid and any associated proteins andpolypeptides, extra- or intra-cellular or transmembrane, leading topolymerization and cross-linking.

Embodiments of the present invention include naphthalimide-substitutedbiomolecules. The naphthalimide compound may be a4-amino-1,8-naphthalimide or a modified naphthalimide, such as Bradsyl.The biomolecule may be chitosan or another macromolecular species. Thenaphthalimide-substituted biomolecule may be in gel form and within acompatible pH range. When irradiated with light in the 400-500 nmabsorption range, the species forms a chemically-reactive species that,upon contact, bonds connective tissues together and bonds collagenousbiomaterial together and to other connective tissues. The biomolecularchitosan moiety of the photochemical may favorably provide anenvironment which stabilizes and protects the reactive species, derivedby the photochemical reaction, until contacted with a connective tissuesubstrate. Thus, the naphthalimide-labeled biomolecule is capable ofbonding tissues with or without subsequent irradiation of the contactedtissue area.

One embodiment of the present invention, in which thechemically-reactive tissue bonding species is formed in the absence ofthe tissue substrate and is sufficiently long-lasting, obviates the needfor direct illumination of the photochemical covered tissue surfacesduring bonding. The naphthalimide-labeled biomolecule acts as anadhesive which allows controlled delivery of the tissue-bonding compoundand facilitates bonding in the absence of excessive compression. This isessential in vascular applications, in which it is imperative to avoidintraluminal bonding which could result in obstructed blood flow. Thepresent invention also provides immediate hemostasis and promotesprimary healing in the absence of excessive proliferation orinflammation. The use of the naphthalimide-labeled biomolecules of thepresent invention also has the potential to reduce operative times.

Naphthalimide compounds of the present invention are useful for varioustissue bonding applications, including vascular patch repairapplications, and for constructing three dimensional objects frombiomaterials, such as prostheses or grafts. Furthermore, thenaphthalimide compounds of the present invention may be sterilized bystandard steam autoclaving for safe biological use without losing theability to bind tissue.

Another embodiment of the present invention relates to methods forstabilizing the expanded shape of a dilated vessel wallpost-angioplasty. Delivery of a naphthalimide compound to an expandedarterial region, followed by activation by an activating agent, caninitiate cross-linking of proteins within the arterial wall and causethe post-angioplasty configuration of the lumen to be maintained. Use ofthe naphthalimide compounds within the expanded artery preferablycreates a relatively smooth vessel lumen, limiting activation of thecoagulatory process and thrombus formation which might otherwise resultfrom healing of the intimal and medial arterial dissections. Theproximity of the tissue bond is determined by the length of thestructural bridge, or spacer moiety, between the two reactivenaphthalimide rings. Such close apposition limits exposure ofsubendothelial elements to circulating blood and vasoactive factorsassociated with the restenosis process. Replacement of the presentlyused metallic or polymer stents with this endogenous, non-metallic“stent” would favorably eliminate the post-operative problems associatedwith these implanted devices and would reduce device costs.

A further embodiment of the present invention relates to methods fordelivering a pharmaceutical agent to a targeted site on a tissuesurface, such as an arterial wall. A pharmaceutical agent, such as ananti-restenotic agent, can be photochemically anchored to the arterialwall through a covalent linkage between the pharmaceutical agent thenaphthalimide compound at an inert site, thus preserving the biologicalactivity of the agent. The naphthalimide compound is thenphotochemically activated by an activating agent and linked to collagenand other proteins in the arterial wall. Linkage of the pharmacologicalagent to the tissue site limits reperfusion washout. Cleavage of thetether will release the pharmacological agent for potential cellularinteraction, if this is desired. This photochemical tethering of apharmacological agent to targeted tissue areas may be used inassociation with various applications to provide local delivery ofdrugs, skin care products, sunscreens, and cosmetics to the skin and tomany other anatomical, physical, surgical, or medical sites.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 is a generalized representation of the labeling of chitosan witha naphthalimide compound (Bradsyl).

FIG. 2 shows four representative structures (I)-(IV) of non-azo4-amino-1,8-naphthalimide compounds.

FIG. 3 is a representation of the process for tethering heparin to atissue substrate using a naphthalimide compound.

FIG. 4 shows two example sunscreen compounds having sulfonic acidfunctional groups.

FIG. 5 shows an example of a process used to attach a sunscreen compoundhaving a sulfonic acid functional group to chitosan.

FIG. 6 shows an example of a chitosan backbone having a sunscreencompound and a naphthalimide compound covalently attached.

FIG. 7 shows six example sunscreen compounds having alcohol functionalgroups.

FIG. 8 shows four example sunscreen compounds having amine functionalgroups.

FIG. 9 shows an example of a process used to attach a sunscreen compoundhaving an alcohol or an amine functional group to chitosan.

FIG. 10 shows the effects of various amounts of light activation on bondstrengths between pericardium tissue samples bonded with anaphthalimide-labeled compound.

FIG. 11 shows the effects of various amounts of light activation andcompression on bond strengths between pericardium tissue samples bondedwith a naphthalimide-labeled compound.

FIG. 12 shows the bond strengths with various amounts of compressionbetween pericardium tissue samples and sections of carotid artery andthoracic aorta.

FIG. 13 shows the viability of vascular smooth muscle cells exposed tosupraphysiological doses of chitosan and a naphthalimide-labeledcompound.

FIG. 14 shows the viability of human umbilical vein endothelial cellsexposed to supraphysiological doses of chitosan and anaphthalimide-labeled compound.

FIG. 15 shows the cross-sectional profiles of viable arterial segmentssubjected to simulated repair of post-angioplasty vascular injury andstabilization of the expanded arterial diameter by a naphthalimidecompound.

FIG. 16 shows the uptake and retention of a hydrophilic naphthalimidecompound in samples of arterial wall based on different delivery methodsand after being washed out.

FIG. 17 shows the uptake and retention of a lipophilic naphthalimidecompound in samples of arterial wall based on different delivery methodsand after being washed out.

FIG. 18 shows a comparison of the uptake and retention of hydrophilicand lipophilic naphthalimide compounds in samples of arterial wall basedon different delivery methods and after being washed out.

FIG. 19 shows an example of a dimeric hydrophilic4-amino-1,8-naphthalimide compound.

FIG. 20 shows an example of a naphthalimide-labeled biomolecule.

FIG. 21 shows an example of a dimeric lipophilic4-amino-1,8-naphthalimide compound.

FIG. 22 shows examples of three isomers (A, B, and C) of a dimerichydrophilic 4-amino-1,8-naphthalimide compound.

FIG. 23 shows an example of a monomeric hydrophilic4-amino-1,8-naphthalimide compound.

FIG. 24 shows the adherence of a sunscreen-modified biomolecule to skinover time.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

One aspect of the current invention pertains to naphthalimide compoundsand their use in tissue bonding and protein cross-linking applications.The naphthalimide compounds can be activated in the simultaneouspresence of an activating agent and a target tissue or protein, causingthe naphthalimide compounds to become an adhesive agent and initiateprotein cross-linking. The naphthalimide compounds may be linked tobiomolecules, such as chitosan, creating naphthalimide-labeledcompounds. These naphthalimide-labeled compounds may also be activatedby an activating agent prior to contact with tissue and carry outsubsequent tissue bonding under “dark” conditions, or in the absence ofdirect tissue illumination.

The naphthalimide compounds are useful for tissue bonding, and inparticular, for applications such as arterial repair and stabilizationof an expanded arterial wall. In addition, the naphthalimide compoundscan be linked with pharmaceutical agents, providing targeted delivery ofthe pharmaceutical agents to tissue surfaces.

As used herein, the word “dye” is interchangeable with the word“compound,” as referred to non-azo 1,8-naphthalimides. See U.S. Pat.Nos. 5,235,045; 5,565,551; 5,766,600; 5,917,045; and 6,410,505; thecontent of each of these patents is incorporated by reference herein.

A “non-azo compound” or dye is one that does not possess a functionalgrouping having two nitrogen atoms connected by a double bond.

A “nucleofuge” is any group which can be displaced from a molecule by anucleophile. Examples of nucleofuges include halogens, sulfonate esters,and quaternary ammonium salts.

As used herein, the words “unsatisfied valences” mean less thantervalent. Thus, any nitrogen atom which is less than tervalent ortri-coordinate contains unsatisfied valences.

The “activating agent” as used herein denotes a means or an agent thatis capable of activating, exciting, or sensitizing a photoactivecompound. The activating agent can be radiated energy, electromagneticenergy, laser, electric current, electrons, thermal neutrons orchemicals. The electromagnetic spectrum can include visible light, xenonlight, laser light, near infrared and ultraviolet light. The laser orother radiation energy can be continuous or pulsed. The pulsed energyused is such that the energy supplied has a multiple number of shortpulses of relatively high energy, but at the same time, has a much loweraverage energy rate, The laser could be a Helium-Cadmium laser, argonion laser, a solid state laser, a gas discharge laser, krypton laser,argon ion pumped dye laser, or hollow cathode metal vapor laser orsemiconductor diode laser, and others. Even sources such as conventionalfilament lamp source with appropriate filtering, an arc lamp source withappropriate filtering, even a pulsed xenon flash lamp with appropriatefiltering could be used, or light emitting semiconductor such as GaN andZnSe diodes.

The term “body tissue” as used herein is to be understood to include“body fluid,” red blood cells, white blood cells, platelets, cryoprecipitate from blood plasma, other plasma proteins, bone marrow, skin,blood vessel wall, nerve sheath, meniscal cartilage, fermoral articularcartilage, cornea, ligament, tendon and other tissues from an animal ora human.

The term “animal” as used herein is to denote any animal; this includeshuman and other domestic and farm animals.

The term “carrier” as used herein denotes a vehicle, a solutioncontaining water, buffers, serum, serum proteins, lipoproteins,artificial bio-membranes, micelles, liposomes, monoclonal antibodies,carbohydrates, cyclodextrans, organic solvents or other pharmaceuticallyacceptable, or compatible, solutions. The carrier, or vehicle, used ispharmaceutically compatible in that it is relatively non-toxic to thenormal cells and normal tissues and it does not react with the solute ortherapeutic agent contained therein.

The phrase “effective amount” as used herein is to denote theconcentration or level of the compound that can attain a particular end,such as cross-linking, without producing pronounced toxic symptoms.

The term “derivative” as used herein is to denote a compound that isderived from some other base compound and usually maintains the generalstructure of the base compound.

In general, the covalent reactions initiated by the activated form ofthese dyes can result in chemical alteration of amino acid residues, ofprotein and peptide conformation and function, and can cross-link theamino acid residues, peptides, and proteins. Thus, this class of dyescan be used to link desired molecular and biomolecular species topeptides, proteins, cells, and biological tissues as well as otherphysiological substrates containing nucleophilic or other reactivegroups, and to cross-link peptides, proteins, tissues, and othersubstrates containing nucleophilic or other reactive groups selectivelyupon application of an activating agent, such as electromagneticradiation with wavelength corresponding in absorption spectrum of thedye absorption spectrum. In addition, graft or prosthetic materialscontaining nucleophilic or other reactive groups can be linked to theactivated naphthalimide. See, U.S. Pat. No. 5,235,045. The appropriateelectromagnetic radiation absorption spectrum includes the ultravioletthrough visible light to near infrared and the K-alpha, etc., X-rayabsorption energies of the molecular halogen substituent. Otheractivating agents include thermal neutrons which could be used toactivate boron-containing 1,8-naphthalimide dyes.

The partitioning of non-azo 1,8-naphthalimide dyes into hydrophobic orhydrophilic regions of a tissue, and the capability of activatingcovalent chemical reactions with nucleophilic amino acid residues allowscross-linking of peptides or proteins located either extra- orintra-cellularly or associated with the bilayer membrane selectivelyupon activation. No photochemical cross-linking occurs until the dye hasbeen activated by an activating agent, such as light.

One embodiment of the present invention is a species of compound inwhich a naphthalimide compound is coupled to a biomolecular moiety. Thenaphthalimide is attached covalently via the 4-amino moiety through aphotochemically inert carbon species chain at the deacylated aminospecies positions on the chitosan monomers. The photochemical reactionof the naphthalimide species yields a tissue chemical species whichreacts covalently with biological connective tissue chemical species.Without wanting to be bound by theory, the biomolecular moiety providesan environment which stabilizes and protects the reactive species untilcontacted with a connective tissue substrate. Preferably, thenaphthalimide compound is 4-amino-1,8-naphthalimide and the biomolecularmoiety is chitosan.

The naphthalimide-labeled biomolecule may be represented as:

D-B

wherein D is the naphthalimide compound or molecule or dye and B is thebiomolecule.

Any suitable biomolecule B can be labeled by the naphthalimide compoundsin accordance with the present invention. In particular, biomoleculeswhich may be labeled by the naphthalimide molecule or compound include,but are not limited to, chitosan, protein, hydrolyzed protein, andcarbohydrates. Preferably, the labeled biomolecule is chitosan. Themacromolecular chitosan should be present in partially deacylated form.Chitosan is derived from the deacetylation of chitin. The macromolecularchitosan used should be at least 70% deacetylated, more preferably 85%or more deacetylated.

In a preferred embodiment, the naphthalimide compound or molecule D is a4-amino-1,8-naphthalimide compound represented as any of the fourstructures (I)-(IV) shown in FIG. 2, wherein R, R′, and Q arestraight-chain or branched chain alkyl groups having from 2 to 200carbons, optionally having one or more ether, amide, or amine groups,and X is hydrogen, a halogen, a sulfonate ester, or a quaternaryammonium salt. The structures (I)-(IV) may represent hydrophilic orlipophilic naphthalimide compounds. When substituent X is hydrogen, thecompound is hydrophilic. When substituent X is a nucleofuge, such as ahalogen, a sulfonate ester, or a quaternary ammonium salt, the compoundis hydrophobic or lipophilic. The biomolecule B is preferably linked tothe naphthalimide compound at one of the 4-amino groups, or at an end ofone of the R or R′ groups.

In another preferred embodiment, the naphthalimide compound D has thestructure:

-   -   which is a mixture of stereoisomers, wherein:    -   n is an integer between 1 and 20;    -   R is selected from the group consisting of CH₃, C₄H₉, C₆H₁₃,        (CH₂)₂N(CH₃)₃+, CH₂COOH, (CH)₂CH₂(CH₃)₂COOH, and        (CH)₂CH₂(CH₃)₂COOCH₃; and    -   R* is a bond between D and B.

A preferred embodiment is also directed to a biomolecule that has beenlabeled with a modified naphthalimide called Bradsyl. Bradsyl chloride,a dark reactive naphthalimide, is a fluorescent tag structurally similarto Dansyl chloride and having an IUPAC name of(4-Chlorosulfonyl-phenyl)-carbamic acid2-(2-butyl-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinolin-6-ylamino)-ethylester. Labeling chitosan with Bradsyl results in Bradsyl chitosan, amolecule with improved bonding characteristics. FIG. 1 shows thestructure of Bradsyl, as well as a generalized representation oflabeling chitosan with Bradsyl. A rigid hydrophobic spacer (an aromaticring) places the naphthalimide away from the chitosan backbone, whilethe hydrophobic terminal butyl group tends to further pull thenaphthalimide into hydrophobic environments. Without wanting to be boundby theory, this tendency likely maximizes naphthalimide interaction withhydrophobic tissue environments and productive photochemical reactionsites within the tissue, promoting bonding at the tissue-fillerinterface and yielding high bond strengths with a minimal amount ofcompression.

Bradsyl and Bradsyl derivatives may be used to label differentbiomolecules. One skilled in the field can vary the structuralcomponents of the Bradsyl molecule to tailor the properties to thedesired results. The spacer between the naphthalimide and the darkreactive group of Bradsyl can be varied in length and in character.Longer and shorter alkanes can be attached to the bottom of thenaphthalimide. The neutral hydrophobic end can be changed to a groupwith a positive or negative charge, or to a hydrophilic end with orwithout a positive or negative charge. Finally, other derivatives canutilize amino acids or derivatives of amino acids. The labeledbiomolecule can be varied as well. In preferred embodiments, Bradsylchitosan was synthesized by using Bradsyl to label chitosans. TheBradsyl chitosan compound may be referred to alternately asBradsyl-modified chitosan, Bradsyl-labeled chitosan, or Bradsyl-labeledacid soluble chitosan.

A preferred embodiment of the present invention is directed to thebonding of natural biomaterials containing protein, such as connectivetissue, as well as synthetic materials. Naphthalimide-labeledbiomolecules of the present invention are capable of bonding theseproteinaceous substrates with or without subsequent irradiation of thetissue sections contacted with the adhesive gel. Thenaphthalimide-labeled compounds may be “activated” prior to contact withthe substrate through irradiation or ambient light. Preferably, thenaphthalimide-labeled compounds are first irradiated with blue light inthe wavelength range of about 400 nm to about 500 nm. The “activated”naphthalimide-labeled compounds are then applied to the substratesections to be bonded. Preferably, minimal compression of at least about0.025 kg/cm² is applied to the substrate sections for at least about oneminute and more preferably for at least about five minutes. Additionalirradiation is not necessary, eliminating the need for directillumination of the bonded substrates.

Numerous permutations in the activation protocol (i.e., biomolecule andnaphthalimide compound concentrations, pressure, light intensities, andexposure times) may be used to further enhance overall bond strengthsand improve reproducibility.

In preferred embodiments, the naphthalimide-labeled compounds may beused to bond a body tissue to a proteinaceous substrate, such as anendogenous body tissue, an exogenous biological material, or anexogenous synthetic material. An additional preferred embodiment of thepresent invention is directed to the use of a naphthalimide-labeledcompound in applying a patch for vascular repair. In particular, thenaphthalimide-labeled compound can be used to bond a natural orsynthetic patch substrate to a body tissue, such as the adventitia of anartery, to repair a tissue or arterial defect, using the same procedurefor bonding tissue segments.

A further preferred embodiment of the present invention is directed tothe construction of three dimensional objects from biomaterials byutilizing a naphthalimide-labeled compound to bond portions of thebiomaterials or cross-link proteins and shape them accordingly. Inparticular, the naphthalimide-labeled compound may be used to shapetubular vascular grafts from flat pieces of tissue or to cross-link theelements of a tissue homogenate in suspension to form athree-dimensional object of desired conformation.

Additional preferred embodiments relate to the use of4-amino-1,8-naphthalimide compounds in the creation of an endogenous“stent,” or the stabilization of an expanded arterial wall afterangioplasty, and local drug delivery through the tethering ofpharmacological agents to tissue surfaces via the naphthalimidecompounds. Naphthalimide compounds which may be used in accordance withthese preferred embodiments include those described in U.S. Pat. Nos.5,235,045, 5,565,551, 5,766,600, 5,917,045, and 6,410,505, and U.S.patent application Ser. No. 10/176,824, the content of each of which isincorporated by reference herein. These naphthalimide compounds areparticularly useful for applications involving stabilizing expandedarterial diameters and local drug delivery.

Preferably, the naphthalimide compound should be a non-azo4-amino-1,8-naphthalimide. The naphthalimide compound can be monomeric,dimeric, hydrophilic, or lipophilic. The naphthalimide compound may haveone of the structures (I)-(IV) shown in FIG. 2, wherein R, R′, and Q arestraight-chain or branched chain alkyl groups having from 2 to 200carbons and optionally having one or more ether, amide, or amine groups.The structures (I)-(IV) may represent hydrophilic or lipophilicnaphthalimide compounds. When substituent X is hydrogen, the compound ishydrophilic. When substituent X is a nucleofuge, such as a halogen, asulfonate ester, or a quaternary ammonium salt, the compound ishydrophobic or lipophilic.

A preferred embodiment of the present invention is directed to the useof naphthalimide compounds in stabilizing the diameter of an expandedarterial wall. The naphthalimide compound should be infused into thearterial wall after balloon inflation has deformed the wall and enlargedthe vessel lumen. Light irradiation through the transparent balloonwall, such as by a fiber optic delivered within the balloon, theneffects the formation of an endogenous “stent” by cross-linkingendogenous plaque and wall proteins. With protein cross-linkingoccurring in the dilated state, the post-angioplasty configuration ofthe lumen is maintained. The proximity of the tissue bond will bedetermined by the length of the structural bridge between the tworeactive naphthalimide rings. Molecular lengths of 6 to 50 angstromshave been prepared as simple naphthalimide molecules. Longer moleculesmay be synthesized by attaching the naphthalimide groups to appropriatemacromolecules.

There are many benefits of using the naphthalimide compounds inaccordance with the present invention to produce an endogenous “stent,”compared to traditional angioplasty. After angioplasty, the artery isstretched and the endogenous plaque is fractured and released at theshoulders, exposing the subendothelium to circulating vasoactive factorsand cytokines. However, with traditional angioplasty, the arteryundergoes restenosis with elastic recoil to its original dimensions,with neointimal formation in response to injury that further reduces theluminal area. By maintaining the post-angioplasty configuration whiletacking down intimal flaps, there is less elastic recoil, maintainedcompression of media and plaque, and limited neointimal formation,resulting in increased luminal area.

A further preferred embodiment of the present invention is directed tothe use of naphthalimide compounds for local delivery of any suitablepharmacological agent to tissue or artery regions. In particular, apreferred embodiment encompasses local delivery of pharmacologicalagents to the arterial wall luminal surface following coronary balloonangioplasty, to reduce restenosis. The naphthalimide compounds may beused to not only cross-link intramedia proteins and stabilize thedilated arterial wall, but also to link anti-restenotic agents totargeted components of the arterial wall immediately following balloonangioplasty. Anti-restenotic drugs such as heparin, taxol, sirolimus,and other suitable pharmacological agents, may be tethered to thearterial wall via the naphthalimide compounds. The naphthalimidecompounds may also be used to tether pharmacological agents to tissuesat other anatomical, physical, surgical, and medical sites to treatvarious conditions.

A preferred device for achieving local tethering of the anti-restenoticdrugs to the arterial wall ideally includes a multi-functional vascularcatheter with balloon dilation capability, the capability to deliver theanti-restenotic agent in aqueous medium to sites along the contactinterface between the expanded balloon and luminal surfaces, an opticalfiber with its tip located in the balloon axis which can emit lightuniformly over the balloon-contacted arterial surface for photochemicalactivation, and a perfusion channel to maintain blood flow through thedevice to regions beyond the balloon site.

As shown in FIG. 3, an anti-restenotic drug, such as heparin (with mrepeating units), is covalently attached to the naphthalimide compoundby a tether (length n). Subsequent light activation results in thecreation of a reactive site (*) that quickly bonds with adjacent tissuesubstrates. This provides a means by which to anchor the heparinmolecule within the arterial wall. This will minimize the loss ofheparin, or other potential pharmaceutical agents, to reperfusionwashout, resulting in enhanced local drug delivery. By subsequentnatural, endogenous cleavage of the tether, the pharmacological agentcan be released for potential cellular interaction, if this is desired.Through the nature and identity of chemical species comprising thetether linkages (e.g., polypeptide, polyester, etc.), the rate ofcleavage of the tether by simple hydrolysis and other enzymatic cleavagecan be modulated, thus controlling the rate and duration of drugdelivery. Retention in linked form may be desirable for somepharmacological agents.

A further preferred embodiment of the present invention is directed tothe use of naphthalimide compounds for local delivery and tethering ofdrugs, skin care materials, sunscreens, and cosmetics to the skin. Toaccomplish this, a biomolecule which has been labeled with anaphthalimide compound is also modified with an additional compoundhaving the desired functional property. After physical contact with theskin, the modified biomolecule and its attached functional compound aretethered to the tissue surface. In this way, compounds such as sunscreencan be locally delivered and tethered to the skin to increase theirresidence time, their resistance to water, perspiration, and rubbing,their coverage, and their effectiveness.

The product can be represented as:

D-B-F

wherein D is a naphthalimide molecule, B is a biomolecule, and F is afunctional molecule of a pharmacological agent, a skin care material, asunscreen, such as a UV blocker, or a cosmetic.

In particular, there are three preferred examples of types of sunscreencompounds which can be linked to naphthalimide-labeled biomolecules andtethered to the skin: compounds containing sulfonic acid functionalgroups, compounds containing alcohol functional groups, and compoundscontaining amine functional groups.

Preferred examples of sunscreen compounds containing sulfonic acidfunctional groups include phenylbenzimidazole sulfonic acid andsulisobenzone, which are illustrated in FIG. 4. The sulfonic acidfunctional group is used to link these compounds to a biomolecule suchas chitosan. In one specific example, as shown in FIG. 5,phenylbenzimidazole sulfonic acid is converted to the sulfonyl chloridederivative, which is directly linked with the primary amine group ofchitosan to give the linked sunscreen compound. The naphthalimidemolecule or compound, although not shown in FIG. 5, is also linked tothe biomolecule. FIG. 6 shows a representative example of the covalentattachment of both a sunscreen agent and a naphthalimide to a schematicrepresentation of a chitosan backbone. The sunscreen agent, or otherfunctional compound, and the naphthalimide molecule may be attached ineither order or simultaneously. Approximately one naphthalimide compoundper one hundred sugar monomer units is preferred, but that ratio may behigher or lower. Approximately one sunscreen agent per ten sugar monomerunits is preferred for effective sunscreen protection, but that ratiomay be higher or lower to provide the effective sunscreen protectionprofile desired.

Preferred examples of sunscreen compounds containing alcohol functionalgroups include triethanolamine salicylate, homosalate, dioxybenzone,oxybenzone, octyl salicylate, and avobenzone, as illustrated in FIG. 7.Preferred examples of sunscreen compounds containing amine functionalgroups include padimate O, menthyl anthranilate, octocrylene, andp-aminobenzoic acid, as illustrated in FIG. 8. Compounds in both ofthese groups can be attached to succinyl chitosan by the formation ofeither an ester or an amide functional group. In a specific example, asshown in FIG. 9, chitosan is modified by succinic anhydride to formsuccinyl chitosan. The carboxylate formed would be converted into anester or amide using an activating agent such as EDC. The naphthalimidecompound, although not shown in FIG. 9, is also linked to thebiomolecule.

Example 1 Synthesis of Bradsyl

The first step involves the synthesis of4-(2′-aminoethyl)amino-N-butyl-1,8-naphthalimide. To a solution of4-bromo-1,8-naphthalic anhydride (2.30 g, 9.9 mmol) in ethanol (100 mL)was added 1-butylamine (0.73 g, 10.0 mmol). The mixture was stirred at68° C. for 24 hours. The general reaction scheme is shown below.

Next, 2-aminoethanol (6.0 g, 100 mmol) was added. Heating was continuedfor a further 48 hours, after which the solvent was removed byevaporation under reduced pressure. Recrystallization of the resultantyellow solid from toluene afforded4-(2′-aminoethyl)amino-N-butyl-1,8-naphthalimide (1.75 g, 56%) as yellowcrystals, with a melting point of about 128-132° C. The general reactionscheme is shown below.

Experimental data for 4-(2′-aminoethyl)amino-N-butyl-1,8-naphthalimide:ν_(max) (cm⁻¹): 3350-2800 (br., N—H str.). 1685 (C=0), 1640 (C=0), 1587,1359, 782. ¹H NMR (CDCl₃): δ 8.59 (d, J=7.4 Hz, 1H, C7-H), 8.47 (d,J=8.4 Hz, 1H, C2-H), 8.19 (d, J=8.4 Hz, 1H, C5-H), 7.63 (d, J=8.4 Hz, ofd, J=7.4 Hz, 1H, C6-H), 6.72 (d, J=8.4 Hz, 1H, C3-H), 6.17 (br. t, 1H,NH). 4.17 (d, J=7.3 Hz, of d, J=7.7 Hz, 2H, CH₂—N), 3.43 (d, J=6.1 Hz,of t, J-5.3 Hz, 2H, NH—CH₂), 3.19 (d, J=6.2 Hz, of d, J=5.1 Hz, 2H,CH₂—NH₂), 1.35-1.8 (m, 6H, CH₂CH₂CH₃, NH₂), 0.97 (t, J=7.3 Hz, 3H, CH₃).m/z: 313 (M+H+1, 23), 312 (M+H; 100), 295 (31), 281 (25), 268 (51), 238(24), 224 (63).

The next step generated Bradsyl, or4-(2-[{4-chlorosulfonylphenyl}-aminocarbonyloxy]ethyl)amino-N-butyl-1,8-naphthalimide.To a solution of 4-(2′-hydroxyethyl)amino-N-butyl-1,8-naphthalimide(1.70 g, 5.4 mmol), prepared above, in anhydrous THF (50 mL),p-chlorosulfonylphenyl isocyanate (2.18 g, 10 mmol) was added. Theresultant mixture was stirred under a nitrogen atmosphere for 24 hours,and the mixture was then vacuum filtered to afford the urethane as ayellow solid. The general reaction scheme is shown below.

Experimental data for4-(2-[{4-chlorosulfonylphenyl}-aminocarbonyloxy]ethyl)amino-N-butyl-1,8-naphthalimide:ν_(max) (cm⁻¹): 3426, 3299 (N—H str.), 3195, 3121, 3058, 2970, 2868 (C—Hstr.), 1741, 1703 (imide C=0 str.), 1636 (urethane C=0 str.), 1593,1551, 1470, 1357, 1174, 777. ¹H NMR (DMSO-d₆): δ 9.85 (br. s, 1H,NHCO₂), 8.73 (d, J=8.4 Hz, 1H, C7-H), 8.44 (d, J=7.0 Hz, 1H, C5-H), 8.29(d, J=8.4 Hz, 1H, C2-H), 7.3-7.8 (complex m, 5H, ClSO₂C₆H₄, C6-H), 6.89(d, J=8.5 Hz, 1H, C3-H), 4.41 (br. t, J=4.6 Hz, of d, J=7.3 Hz, 2H,O—CH₂), 4.01 (t, J=6.7 Hz, 2H, N—CH₂Pr), 3.71 (br t, J=4.6 Hz, 2H,CH₂NH).

Example 2 Synthesis of Bradsyl-Labeled Chitosan

To synthesize the Bradsyl-labeled chitosan compound, the chitosan ispreferably solubilized in an acid such as acetic acid or lactic acid.For example, 2 g of chitosan (Sigma, St. Louis, Mo.) and 40 mL of 10%acetic acid were combined and the chitosan was allowed to solubilizeovernight.

Next, 100 mg of the naphthalimide, such as Bradsyl, was dissolved in 1mL of acetone or DMSO. The naphthalimide solution was then added slowly,with stirring, to the chitosan solution. If it is added too quickly, thechitosan will precipitate. Immediately after adding the naphthalimide, 6M KOH was added slowly, with stirring, until the pH of the solution wasabout 8 to about 9 as tested by pH paper. If the target pH was exceeded,back titration was not performed. The pH was monitored over the courseof about 3 to about 4 hours, adding more base as necessary to keep thepH between 8 and 9. The mixture was allowed to stir at this pHovernight.

Then, 100 mL of 10% acetic acid was added. The solution was stirreduntil the modified chitosan was completely dissolved, or for at leasttwo hours. The solution was centrifuged and any insoluble material wasdiscarded. The insoluble material consists of excess naphthalimide andinsoluble or overmodified chitosan.

To purify the solution by alkaline precipitation, 6 M KOH was added tothe supernatant until the pH was about 8 to about 9. The modifiedchitosan was allowed to precipitate for at least thirty minutes. Theprecipitate was then collected via centrifugation.

Finally, the modified chitosan solution was added to dialysis tubingwith a syringe and both ends of the tubing were tied off. The dialysistubing was placed in a 1 L Erlenmeyer flask and about 1 L of 10% aceticacid was added. The sample was dialyzed until all of the chitosan hadresolubilized, or for at least 12 hours. The dialyzate solution wasdiscarded after noting the color and replaced with 1 L of deionizedwater. After 12 hours the dialyzate solution was replaced with freshdeionized water and was dialyzed for an additional 12 hours. Finally thesample was dialyzed against phosphate buffered saline (“PBS”) for 24hours.

The concentration of chitosan in the homogenous modified mixture wasdetermined by taking an aliquot, typically 1 mL, and determining theweight. This sample was then dried and the residue weighted. Theresulting data (mass chitosan/mass sample) yielded the concentration ofsolids, typically expressed as mg/g. Samples with chitosanconcentrations between 1 and 100 mg/g have shown effective bonding.Samples with chitosan concentrations between 10 and 50 mg/g have shownthe greatest efficacy.

The modification ratio of Bradsyl Chitosan was determined by taking a1.00 mL aliquot and diluting it to 25.00 mL with 10% acetic acid. Theoptical absorbance was measured at 450 nm. After correction fordilution, a molar extinction coefficient of 20,000 M⁻¹cm⁻¹ was used todetermine the naphthalimide concentration in the sample. The mass ofchitosan in the sample was used to determine the concentration of sugarsubunits, using a molar equivalent weight of 180 g/mole. Themodification ratio was expressed as a ratio of sugar subunits to boundnaphthalimide groups. A larger number indicates a lower level ofmodification. A Bradsyl chitosan with a modification ratio of at least1500 (1 naphthalimide per 1500 sugars) has been shown to be effective intissue bonding. Better results are obtained with a modification ratio ofat least 500. The best results are obtained with a modification ratio ofat least 100.

Example 3 Synthesis of Labeled Chitosan

Standard amide coupling reactions were used to attach variousnaphthalimides to chitosan.

Two grams of chitosan (Sigma) were dissolved in 40 mL of 8% acetic acid.This solution was diluted with 160 mL of methanol and treated with 1.4 gof succinic anhydride dissolved in 50 mL of acetone. This succinylatedchitosan is purified by repeated basic precipitation followed bysolubilization in 0.1 M HCl. The naphthalimide was attached to thechitosan using cardodiimide-mediated coupling reactions. A saturatedchitosan in 0.1 M HCl solution was diluted five-fold with methanol andan amine terminated naphthalimide (having the structure shown in FIG.19) was added to this solution. The addition of a coupling reagent(N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride) resultedin the covalent attachment of the naphthalimide. This modified chitosanwas purified by either repeated extractions with DMF or by repeatedbasic precipitation followed by solubilization in 0.1 M HCl.

Example 4 Cross-Linking of the Naphthalimide-Chitosan Species

Studies were performed to determine if cross-linking occurred within thenaphthalimide-chitosan gel after photoactivation. The gel was formed bysolubilizing chitosan (Biopolymer Engineering, Inc., Eagan, Minn.) inPBS at 25 mg/mL, then making a 1:4 dilution. Free naphthalimide compound(having the structure shown in FIG. 19) was added to make an 8 mMconcentration. Next, 20 μL aliquots of this solution were dispensed onclean glass microscope slides. A group of these slides were then exposedto activating blue light (800 mW/cm²) for 7 minutes. A control group ofslides was maintained in the dark until air-dried. 200 μL of H₂OD wasused to rehydrate the solutions. While all of the controls quickly wentback into solution, the specimens containing the naphthalimide andexposed to the activating blue light formed distinct and durable filmswhich did not redissolve in water.

The films of the treated specimens were also subjected to agitation,acetate buffer, 1% SDS, and H₂OD at pH 4 and 6. The cross-linkedchitosan films maintained their configuration even after a week orlonger in the various solutions. Observed under the microscope, thesefilms had a very distinct appearance with crystalline-like features. Thecontrol specimens did not show these features.

Aggregate formation was also detected chromatographically in anaphthalimide-modified chitosan solution that had been exposed to roomlight only, rather than a filtered arc lamp light source. Liquidchromatography produced three distinct peaks. The peak at about 1500seconds was indicative of native chitosan materials, while the peaks atshorter time periods represented larger aggregate molecules formed bynaphthalimide-induced photochemical cross-linking.

Example 5 Effects of Light Exposure on Tissue Bonding

An example of the naphthalimide-labeled chitosan (having the structureshown in FIG. 20) also demonstrated an ability to bond tissues under“dark” conditions, or having been exposed only to room light duringsynthesis and procedures, rather than blue light irradiation. FIG. 10shows the results using various amounts of light activation. Thenaphthalimide-labeled compound was designated RXN1.

A screening model of swine pericardium was used. The pericardium hasbeen identified as a potential biological patch material for arterialrepair. This highly collagenous tissue is thin and fairly translucent,therefore optimizing the potential for light penetration, while alsobeing durable and readily available.

A thin film of the experimental naphthalimide-chitosan formulation (20μL) was applied to the pericardium. Some compound samples were preparedentirely in red light, a wavelength at which there is no absorption bynaphthalimide, and thus were prepared effectively in the dark (D).Others were prepared in ambient laboratory light, and others weresubjected to 5 minutes of blue light irradiation at 800 mW/cm² prior totissue application (L). The pericardial patch segment was then apposedto the treated tissue in an area of overlap with single-thickness“tails” projecting from each end. Some tissue samples were thensubjected to an additional 5 minutes of blue light irradiation (−5). Allsamples were clamped at 5 kg/cm² for 5 minutes. After the tissues werebonded, the tissues were carefully placed in PBS for at least an hourprior to tensile strength testing to ensure any residual “stickiness”resulting from partial dehydration would not influence the measuredtensile strengths. Testing of tensile bond strengths was conducted usingan incremental loading system, whereby the force was gradually increasedto the point of bond failure. Peak loads were noted and bond strengthswere calculated as g/cm².

The bond formed in ambient laboratory light but the absence of arc lampexposure (R1-0) showed significant strength when compared to the bondformed in ambient light with arc lamp exposure (R1-5). In particular,the bond formed in the absence of arc lamp exposure had approximately73% of the strength of the light exposed bond. Exposure of the compoundsto 5 minutes of blue light irradiation at 800 mW/cm² prior to tissueapplication, followed by subsequent clamping at 5 kg/cm² for 5 minuteswithout arc light exposure to the clamped tissues, produced higher bondstrength (LR1-0), approximately 58% greater than that of the bondsprepared in ambient light alone (R1-0). The strongest bonds wereobtained when the compounds were activated prior to application and thetissue was irradiated afterward as well (LR1-5).

Solutions and tissue bonds that were protected from room lightactivation entirely (DR1-0) consistently showed the lowest bondstrengths. However, light exposure of 5 minutes at 800 mW/cm² producedan approximate 50% increase in bond strength (DR1-5).

The results indicate that both incidental lab light exposure andcontrolled blue light exposure prior to application of the bonding gelto the tissue can produce improvements in bond strengths, eliminatingthe requirement that tissues be irradiated during the bonding procedure.

Example 6 Tissue Bonding with Varying Light and Pressure

Bradsyl-labeled chitosan exposed only to ambient laboratory light duringsynthesis and experimental manipulations, was used. The bondingprocedure was substantially the same as that described in Example 5,using alkylated bovine pericardium (Veritas™ sheets, Synovis Surgical,St. Paul, Minn.). Results are shown in FIG. 11. The sample in which noexternal pressure was applied to the lapped joint (tu-5) showed poorbond yield strengths. The addition of the weight of a single glassslide, about 0.025 kg/cm², which ensured apposition of the tissues,substantially improved performance. Mean bond strength nearly doubledbetween one minute and five minutes of this minimal compression.

Blue light irradiation of the tissue samples, ranging from 1.25 to 5minutes at 800 mW/cm², appeared to consistently reduce bond strengthwhen compared to samples with the same compression time. Without wantingto be bound by theory, it is likely that ambient light prior to tissueapplication produces chemical species in the adhesive compound thatcause subsequent bonding in the tissue environment. Additional intenseblue light appeared to destroy these productive bonds or deplete thechemical species.

Example 7 Tissue Bonding in Arterial Repair

The Bradsyl chitosan adhesive was also tested in adhering a bovinepericardial patch (Veritas™, Synovis Surgical, St. Paul, Minn.) to theadventitia or outer surface of the thoracic aorta and carotid artery ofrabbits. The procedure described in Example 3 above was used. One samplewas tested with minimal compression, or with an overlying glass slide toimpose about 0.025 kg/cm² pressure on the bond area for about 5 minuteswithout any direct blue light illumination of the tissue bonding region.Another sample was subjected to moderate compression, by compressing thetissue between two glass slides with rubber bands, which is estimated tobe about 1 kg/cm² pressure. A third sample used a patch overlyingarterial wall sheets opposed as a butt weld. As seen in FIG. 12, withmoderate compression, lap bond strengths of up to about 0.225 kg/cm²were formed for the thoracic aorta and about 0.150 kg/cm² were formedfor the carotid artery. Bond strengths using the reinforced butt weldwere about 0.150 kg/cm² and 0.075 kg/cm² for the thoracic aorta andcarotid artery, respectively. The thoracic aorta bonds were consistentlystronger than those of the carotid artery. Furthermore, moderatecompression appeared beneficial.

While these overall bond strengths were notably lower than thoseobserved in pericardium-pericardium tissue bonds, they still providesignificant adherence of the patch to arterial adventitia. Thus, theresults support the application of the Bradsyl chitosan gel, with lablight exposure, in arterial patching. The application is particularlysupported for smaller arteries, with a diameter of about 3 mm or less,for which tangential wall hoop stress value calculations of less than0.15 kg/cm² from an internal pressure of 300 mmHg is less than thevalues realized in the above patch-arterial adventitia bonds. TheBradsyl chitosan gel is therefore useful in the sutureless repair ofsmaller blood vessels.

Example 8 Cytotoxicity Testing of Bradsyl Chitosan Gel

The primary limitations of current bio-adhesives lie either ininsufficient bond strengths or toxicity issues. While the Bradsylchitosan adhesives demonstrate excellent bond strength andreproducibility, these new formulations were also subjected to toxicitytesting. Cytotoxicity was assessed using a cell culture model. Two celllines were exposed to varying doses of the adhesives. Cell viability wasmeasured using the MTT (tetrazolium salts) spectroscopic lightabsorption based assay. Control wells were also prepared to determinerelative toxicity.

The results, shown in FIGS. 13 and 14 were obtained in cultured vascularsmooth muscle cells (“VSMCs”) (FIG. 13) and endothelial cells (“ECs”)(FIG. 14). These cell lines showed varying degrees of sensitivity to theadhesive when exposed to 40 μL of the test compound in 100 μL of mediafor two days. The 40 μL amount of the test compound was chosen becauseit represents the amount of adhesive used per unit area that is appliedduring the ex vivo tissue bonding protocol. This does not account forrun-off and displacement as the tissues are overlapped and compressed.Furthermore, during tissue bonding, only the surface cells are directlyexposed. Subsequent layers are exposed by diffusion at lower doses.Therefore, the cytotoxicity tests represent the cellular response tosupraphysiological doses with direct cell contact.

The addition of 12.5 mg/mL chitosan gel alone appears to have nodeleterious effects on cell viability. Indeed, chitosan showed atendency to enhance growth, particularly in ECs, with a 354% increaseover the controls. This feature is advantageous in wound repair. TheBradsyl chitosan results were comparable to control wells exposed tomedia alone or 40 μl of PBS in 100 μl media, demonstrating that theformulation is relatively innocuous with regard to cellular toxicity.Because these compounds do not require blue light activation subsequentto tissue application, this variable was not tested. This feature of theBradsyl-labeled compounds preserves tissues from any cell-damaging bluelight toxicities. Thus, even at supraphysiological doses, theBradsyl-labeled compounds demonstrate high bond strengths and apromising biocompatibility profile, which furthers their applications insoft tissue bonding and wound closure.

Example 9 In Vitro and In Vivo Arterial Patching

In vitro patch repairs were performed on euthanized rabbit aortasegments in an organ chamber using Bradsyl-labeled chitosan, underprotocol approved by the University of South Dakota Institutional AnimalCare and Use Committee, and essentially as described in Example 3 above.A puncture wound was made through the arterial wall of the arterialsegments. Following deposition of 20 μL of the adhesive gel to thearterial surface (Veritas™, Synovis Surgical, St. Paul, Minn.), thepatch and arterial surfaces were apposed with gentle molding of thepatch outer surface to conform to the outer cylindrical surface of theinfolded arterial segment. The estimated pressure was approximately0.025 kg/cm². The gentle molding contact was maintained for 5 minutes,with all of the patch surface contacted.

Although bulging of the patch over the puncture wound was noted, patchsegments withstood intraluminal pressures exceeding 1200 mmHg beforeleakage was observed.

In vivo testing of the patch was also performed under the review of theUniversity of South Dakota Institutional Animal Care and Use Committee.After the animals were anesthetized, the abdominal aorta was exposedbetween the renal arteries and the aorto-iliac bifurcation andtemporarily clamped. A forceps was used to pinch and lift the aortawhile small scissors were used to make a 3 mm longitudinal, irregularopening. This was sealed as described in the in vitro study. After 5minutes of bonding time, the clamps were removed, allowing the return ofblood flow and pressure. In all animals tested (n=6), no bleeding wasobserved during the subsequent observation under anesthesia (about 30minutes). Following each experiment the animal was euthanized accordingto the approved protocol.

The results support the use of the Bradsyl chitosan gel in vascularrepair.

Example 10 Fabrication of a Tubular Vascular Graft

The following experiments were designed to explore the application ofthe Bradsyl chitosan technology to fabrication of collagenousbiomaterial prostheses, 3 dimensional shaping, bonding, and sealing of atubular form of pericardium (Veritas™, Synovis Surgical, St. Paul,Minn.). Sheets of the pericardium, approximately 4 cm wide with varyinglengths, were rolled up on a wooden mandril having an outer diameter ofabout 8 mm. This resulted in a tube 4 cm in length with an 8 mm diameterand overlap regions of 10, 20, or 30 mm, to which the Bradsyl-labeledchitosan adhesive was applied. After removal of the mandril, these tubeswere mounted in an organ chamber for subsequent burst pressure testing.Regardless of overlap area, average burst pressures of approximately 250mmHg were observed.

Another approach to the fabrication of a tubular vascular graft involvesthe homogenization of a purified, acellular collagen matrix (Veritas™,Synovis Surgical, St. Paul, Minn.). Preliminary experimentation usedBradsyl chitosan (10 mg/mL) to cross-link Veritas™ homogenate at a 1:1ratio of adhesive to homogenate. This resulted in definitivecross-linking of the homogenate, with the cross-linked materialretaining cohesiveness over the course of 8-12 days in PBS at pH 7.4,while uncross-linked controls completely disassociated upon rehydration.The Bradsyl chitosan technology can be employed to crosslink the tissuehomogenate into desired cylindrical conformations of varying diameters,with or without bifurcations or other prosthetic design.

Example 11 Naphthalimide Compound Penetration of Atheromatous ArterialTissue

The following experiment was performed to analyze the compound form,compound concentration, and compound exposure time that provide adequatetissue penetration for expanded arterial stabilization.

These experiments utilized atherotic carotid artery from young adult(3-6 months) male New Zealand White rabbits. Atherosclerotic lesionswere created using the air-desiccation model (LeVeen, 1982).Approximately 4 cm lengths of both the right and left carotid arterywere harvested from donor animals. These segments were openedlongitudinally and divided into 8 small rectangles, which provided 16test segments from each animal. These arterial pieces were immersed inhigh or low concentrations of a solution of a lipophilic naphthalimidecompound (having the structure shown in FIG. 21) or a solution of ahydrophilic naphthalimide compound (a mixture of the three isomers A, B,and C shown in FIG. 22) for periods of either 5, 15, or 30 minutes. Thelipophilic naphthalimide was dissolved in 20% Cremophor® EL (BASF, MountOlive, N.J.), a lipophilic solvent and micellar agent, to provide anaqueous stock solution (0.9 mM, determined via optical density). Thehydrophilic bis-naphthalimide solution was dissolved in PBS, ahydrophilic solvent (0.9 mM solution). Solvent controls included PBS andCremophor®. “Low” concentrations were made by making a 1:2 dilution ofthe stock solution.

Following incubation in solution, artery segments were frozen in liquidnitrogen, cryosectioned and examined by confocal microscopy. Images wereanalyzed using commercial image analysis software. Fluorescent profilesemitted by the incorporated naphthalimide compounds were used todetermine an average intensity and the depth of compound penetration.Because each animal provided sufficient tissue to contribute to allexperimental groups, a paired statistical design was utilized for dataanalysis. Because of the heterogeneity of variance, the Friedmanstatistic was used, followed by a modified Student Newman Keul'smultiple comparison test.

Analysis of the results showed that immersion of the artery segments inhydrophilic compound, the bis-naphthalimide, resulted in compoundpenetration from the luminal side at all concentrations and exposuretimes tested. After 5 minutes of incubation time the low concentrationhad penetrated approximately 31% of the medial thickness compared to 41%with the high concentration. After 15 minutes, the low and highconcentrations penetrated 54% and 80% respectively. After 30 minutes ofimmersion, the low and high concentrations penetrated 77% and 100%respectively with the high concentration at 30 minutes penetratingbeyond the medial wall into the outer adventitial layer, a cumulativepenetration of 129%.

The lipophilic compound penetration and localization in the atheromatousvascular wall segment differed markedly from the hydrophilic form, withthe former being localized primarily in the adventitial wall component(increasing with time and concentration) rather than within the media.Maintenance of compression of this wall component is unimportant in thestabilization of luminal patency following balloon dilation. Controlsshowed virtually no fluorescence after autofluorescence was filteredout.

The results indicated that the naphthalimide compounds were taken up bythe arterial wall.

Example 12 Tissue Bonding in Atheromatous Arterial Tissue

The following experiment was performed to examine whether, upon adequatecompound exposure and radiation, sufficient molecular interaction wouldoccur between apposed luminal surfaces of atherosclerotic rabbit carotidartery to cause a significant increase in the strength of bonds overcontrols.

Rabbit carotid arteries were lesioned and dissected as described inExample 11 above. Isolated arterial segments were catheterized with a3.0 mm balloon catheter and subjected to a standardized ballooninflation protocol (3 inflations to 6 atm with 30 second inflation rampsand one minute inflations with a one minute rest period betweeninflations). This protocol was used to produce arterial injury that maybe characteristic of that observed post-angioplasty in vivo. Each arterywas opened longitudinally, with each rectangle cut into four pieces,yielding a total of eight tissue segments from each animal. In thismanner, each animal contributed to one experimental and three controlgroups allowing for a paired statistical design.

The experimental group was prepared by immersing the segments inhydrophilic compound solution (same solution used in Example 11, at aconcentration of 0.9 mM) for 30 minutes. Two saturated arterial segmentswere positioned so that the luminal surfaces were apposed in an area ofoverlap, with single thickness “tails” projecting from each end. Thetissue prep was wrapped in a thin polyurethane sheet, sandwiched betweenglass microscope slides, and clamped with thin C-shaped spring steelclips. Based upon spring-load deflection calibration, the resultantpressure was estimated at 3 kg/cm², sufficient to bring tissue in closeapposition. The tissue was then exposed to 400-500 nm wavelength lightfrom a 159W arc lamp for 30 minutes at an intensity of 800 mW/cm². Thedilating force of a polyethylene balloon catheter at 6 atm isapproximately 15.75 kg/cm², which provides sufficient pressure to apposesurfaces within wall and plaque by balloon dilation.

Control groups consisted of atheromatous sheets soaked withnaphthalimide solution, clamped and held in the dark, and sheets paintedwith naphthalimide-free PBS and held in the dark or irradiated. Solutiontemperatures were held at 27° C.

Following light exposure or equal dark holding times, tissue wasunclamped and removed from the polyurethane. Samples were rehydrated insaline prior to testing of tensile strengths (Sintech instrumentation).Pneumatic grips secured the single thickness “tails” of the overlappedtissue segments. The grips were then progressively separatedmechanically to increase tension in the area of overlap. Computergenerated graphs of the stress load yielded the peak stress achievedprior to separation of the apposing surfaces. The Friedman statistic andmodified Student Neuman Keul's multiple comparison statistical testswere used for analysis.

Control groups consistently failed, with values no higher than 0.035kg/cm², while the hydrophilic dimeric 1,8-naphthalimide (MBM Gold12-11-12, MicroBioMed. Corp., Dallas, Tex.) yielded bond strengthsaveraging 0.07 kg/cm² for the arterial wall segments. Subsequentexperimentation with thoracic aorta segments yielded bond strengths of0.122 kg/cm².

The results indicate that cross-linking does occur between proteinconstituents of the arterial wall, which is necessary to maintain theexpanded diameter and repair intimal and medial dissection, thuslimiting a proliferative reparative response and ultimately restenosis.

Example 13 In Vitro Stabilization of Dilated Artery Wall Dimensions

The following experiment used intact cylindrical arterial segmentsperfused in an organ chamber to more closely simulate in vivoconditions. The organ chamber was equipped with a dissecting scope,video camera, and VCR to enable magnified views of the artery and tostore images for later review. The ability of the technique to repairangioplasty-induced intimal and medial dissections was evaluated fromhistological analysis.

Rabbit carotid arteries were lesioned as described in Example 11, exceptthat a more discrete lesion (approximately 1 cm) was produced. After 4-6weeks of plaque development, the carotids were dissected and excised asin Example 11. The isolated 4 cm arterial segment was mounted in aspecialized organ chamber by cannulating both arterial ends with tubingthrough which 37° C. physiological saline solution perfusate wascirculated using a diaphragm metering pump to maintain oxygenatedperfusate solution and nutrient supply. A 95% oxygen/5% carbon dioxidegas mixture was bubbled into the saline solution reservoir to generateoxygenated perfusate. The segment was anchored to maintain its in situlength. Following mounting of the artery the vessel was perfused for 30minutes to allow the arterial tissue to equilibrate. Intraluminalpressures were measured continuously using a computer-based physiograph.

Each arterial segment then underwent a standardized balloon inflation tosimulate PTCA. Balloon diameters were chosen to approximate a 1.3:1.0ratio of maximal balloon diameter to “normal” vessel diameter. Theballoon catheter was introduced into the vessel lumen via a permeableseptum perforated by an introducer/sheath that accommodates the ballooncatheter shaft. The catheter was advanced until the 2 cm balloon bridgedthe center portion of the arterial segment. The standard angioplastyprotocol included three inflations to 6 atm with 30-second inflationramps and 60 second inflations. Each inflation was ended by free releaseof the inflation mechanism followed by 30 seconds to monitor baselinepressures. Video recording was continuous throughout. External diameterchanges were recorded using edge detection software.

After balloon dilation, experimental arteries were filled with 12 mMhydrophilic naphthalimide compound for 30 minutes to ensure diffusion ofthe compound into the tissue. A final single inflation was performed.The arterial segment was irradiated with 800 mW/cm² for a total of 45minutes during balloon inflation, with the artery being rotated twice toensure irradiation of the entire arterial circumference. Intraluminalirradiation via a fiber optic guidewire would improve uniformity oflight delivery and reduce required irradiation times. Technology iscurrently available to provide intraluminal light delivery.

Control groups consisted of a group with no irradiation, a group withnaphthalimide without irradiation, and a group with saline instead ofnaphthalimide compound.

After treatment, the perfusion solution was changed to phosphatebuffered glutaraldehyde, tissues were perfused, and tissues weresubsequently stored in the same solution to fix the tissue for light andelectron microscopic analysis.

Histologic and morphometric analyses consisted primarily of (1)determination of cross-sectional area, medial area, intimal area, andpercent plaque, and (2) determination of luminal circumference andexternal medial circumference and other physical dimensions as well ashistologic evaluation of tissue injury. The morphometric analysesquantified luminal, plaque, and medial areas on perfusion-fixed treatedand control arteries.

Considerable variability was observed in the extent of plaque found inthe segments independent of group. Although this variability mademorphometric analyses, shown in FIG. 15, more difficult, there was astrong trend for medial thinning in the naphthalimide/light group. Themedial thickness represented 5.5%±2.2% of the outer radius versus9.2%±2.2% in the PBS/dark group. Luminal areas tended to be larger inphotochemical and light treated groups.

The results indicate that stabilization of the dilated lumen dimensionalarea by photochemical means does occur, creating an endogenous “stent.”

Example 14 Retention of Compressed Arterial Wall by Cross-Linking

Compression of the atherosclerotic plaque and other tissue componentsshould result in gain in lumen diameter. Example 13 showed medial wallthinning and gain in lumen diameter following photochemical bonding inthe dilated state. Furthermore, previous experiments with compressedoverlapped skin painted with naphthalimide and irradiated showedstabilization of the compressed skin, with the thickness equals to 70%of that of uncompressed skin. The following experiment was done todetermine if similar retention of tissue compression was observed invascular tissue.

Swine coronary arteries were dissected from fresh post-mortem swinehearts. Each artery was opened longitudinally and 3 mm discs wereobtained using a biopsy punch.

The experimental group (naphthalimide/light) was prepared by immersingthe discs into hydrophilic compound solution (15 mM MBM 10-8-10,MicroBioMed. Corp., Dallas, Tex., a mixture of 3 dimeric isomers) for 5minutes. The disc was then wrapped in a polyurethane sheet, sandwichedbetween glass microscope slides, and positioned in a lever devicedesigned to apply controlled force to the slide surface. Weights of 5.3,10.14, and 20.28 kg/cm² were applied. Light at wavelengths 400-500 nmfrom an arc lamp was delivered to the specimen surface at an intensityof 800 mW/cm² for 10 minutes. Control groups consisted of naphthalimidetreated discs with no light irradiation and discs exposed to bufferedsaline solutions with no naphthalimide, either exposed to light or keptin the dark.

Following light exposure or equal dark holding times, the tissue wasremoved from the pressure device and wrapping film and hydrated inphosphate buffered saline for twenty hours prior to final diametermeasurements, to ensure any retention of compression was not due topartial dehydration. Diameter measurements were made prior to anycompression, immediately following treatment (prior to re-hydration) and20 hours after re-hydration. Friedman statistics and Student NeumanKeul's multiple comparison tests were used for data analysis.

The lower compressive force of 5.34 kg/cm² showed significantcompression of the segments and after the 20 hour re-hydration period,the naphthalimide/light treated group showed significantly greaterresidual compression compared to controls (i.e., naphthalimide/lightgroup—19.4±8.4% vs. naphthalimide/dark group—7.1±5.7%). The highercompressive forces proved to be too high for practical use as all higherforces caused wall damage and irreversible wall distortion. The lowerforce used was comparable with values that can be attained with balloondilation.

The results indicate that photochemical cross-linking of vascular wallconstituents during compression can result in a significant retention ofwall compression before and following re-hydration.

Example 15 Intraluminal Delivery of Naphthalimides

In this experiment, intraluminal delivery via catheter and reperfusionwashout of these naphthalimide compounds was evaluated to determinetheir efficacy for bonding the vascular wall.

Local delivery of these 1,8-naphthalimide compounds was achieved using acommercially available Coronary Infusion Catheter. (DISPATCH™, SciMed®,Maple Grove, Minn.). This catheter incorporates inflatable coils whichcreate drug “compartments” that allow drug contact with the arterialwall. Its perfusion capability permits longer drug diffusion timeswithout causing distal ischemia. This local delivery system allows fordwell times comparable to those used in previous in vitro experimentswhere successful compound penetration was demonstrated.

Normal carotid arteries were harvested from euthanized young adult (3-6months) male New Zealand White rabbits for in vitro experimentation. Asmall length of artery was trimmed from each to serve as eitheruntreated (negative) or immersion-soaked (positive) control tissue insubsequent fluorometric analyses. Arterial segments were catheterizedand subjected to a standard angioplasty balloon protocol (described inExample 13) inflated to 6 atm for three one minute inflations whileimmersed in oxygenated physiological saline (PSS) solution at 37° C. Thestandard balloon catheter was then replaced with the DISPATCH™ infusioncatheter, which was inflated to 6 atm pressure. The infusion port wasloaded with 10 mM lipophilic (MBM Yellow 06-06, MicroBioMed. Corp.,Dallas, Tex.) or hydrophilic 1,8-naphthalimide compound (MBM Gold12-11-12, MicroBioMed. Corp., Dallas, Tex.) Three initial short, strongbursts were used to promote uniform filling of the “drug compartments.”Subsequent infusion at 1.59 cc/hr maintained delivery of the compoundover a 30 minute period. To ensure intraluminal delivery, arterialsegments were suspended over a basin and continuously rinsed with salinerather than immersed to eliminate the potential of delivery of compoundfrom any location except from within the lumen. Washout experiments werealso performed in which PSS was perfused at physiological pressures for10 minutes to evaluate compound retention. One of the control segmentswas immersed in the compound and used as a positive control.

All experimental arterial segments exposed to the naphthalimidecompounds showed a yellow staining of the tissue visible by grossexamination. A fluorometric assay was developed to quantify residualcompound presence in the arterial wall. After a brief rinse andblotting, comparable weights of control and experimental tissue werehomogenized (OMNI Tissue Homogenizer, OMNI International, Warrenton,Va.) in 4 mL of 0.9% NaCl. A standard curve was prepared using knowncompound concentrations. 40 μL of each of the unknown homogenates wasadded to 250 μL of 0.9% NaCl. Assays were run in triplicate using an FL500 Microplate Fluorescence Reader to determine the fluorescence of eachsample. Linear regression was then used to plot the standard curve andextrapolate the concentrations (nmol compound/g tissue) of the unknownfor the various treatments. Compound localization within the arterialwall was demonstrated by fluorescence microscopy. The same statisticalsystem used in previous experiments, Friedman's statistic followed bymodified Student Neuman Keul's multiple comparison test, was employedfor data analysis.

The use of the DISPATCH™ catheter resulted in effective luminal deliveryof both hydrophilic and lipophilic naphthalimide compounds. FIG. 16shows the retention of the hydrophilic compound in the arterial wall,FIG. 17 shows the retention of the lipophilic compound in the arterialwall, and FIG. 18 is a comparison of both compounds and treatmentmethods. Zones of higher intensity of compound were noted in associationwith the spaced gaps in the delivery coils, but dye concentration wasobserved along the entire lumen. The total amount of compound deliveredwas different using the hydrophilic and lipophilic forms, yet wasconsistent with distributions determined previously using immersion.Using the hydrophilic form, intraluminal delivery resulted in greatercompound delivery in four of the five segments tested. In the fifthanimal, immersion values were higher than delivery catheter values. Ingeneral, values of 1136±749 nmol compound/g tissue was demonstrated forhydrophilic naphthalimide compounds delivered using the DISPATCH™ localdrug delivery catheter system.

Since no light exposure was used to bond the tissue during theexperiment, washout (W/O) of the compound was substantial for bothlipophilic and hydrophilic forms. After 10 minutes of saline perfusionpost compound delivery, lipophilic compound levels were 48.2%±38% lowerthan with intraluminal delivery alone. Hydrophilic levels were 89.2%±8%lower than pre-washout levels. Nevertheless, remaining levels weresufficiently high enough to permit direct observation of tissuefluorescence and color.

The results indicate that intraluminal delivery of the photochemical tothe arterial wall components via a DISPATCH™ or similar system toachieve tissue compression stabilization or for drug delivery is asatisfactory system. The hydrophilic compound form is the naphthalimideof choice because it not only localized in regions of the arterial wallbut also photochemically linked proteins within the wall.

Example 16 Local Drug Delivery by Photochemical Tethering

In this experiment, in vitro photochemical tethering of heparin to thearterial wall using naphthalimide was analyzed.

An example of a naphthalimide compound (having the structure shown inFIG. 23) was bound to enoxaparin (Lovenox®, Aventis Pharmaceuticals,Inc., Bridgewater, N.J.) and biotinylated in a 1:1 ratio using standardbiotinylation procedures to provide a means of marking the heparin forhistological localization. The heparin was bound to the naphthalimide asshown in FIG. 3 by covalent attachment to the naphthalimide compound.

This modified heparin compound was then applied to the luminal surfaceof excised swine coronary arteries. Experimental sections wereirradiated with 800 mW/cm² of 400-500 nm blue light for 5 minutes.Control sections received light without the compound, the compoundwithout light, or neither the compound nor light. All sections thenunderwent sequential washings to remove the unattached heparin.Comparisons were made based on the coloration of the sections. Specimensreceiving both the heparin/naphthalimide compound and thephotoactivating blue light irradiation showed strong staining along theluminal surface and, to a lesser degree, throughout the arterial wall.Thus, definitive photochemical attachment of heparin to the wall wasshown with localization primarily on the luminal surface, but withsubstantial penetration and bonding to the media as well. Negativecontrols failed to demonstrate the typical brown staining indicatingresidual heparin presence. All specimens showed some artifactual redstaining within the intima, and the yellowish appearance of thecompound/no light specimen indicated some residual naphthalimidepresence after washings.

Light activation of the naphthalimide, as indicated previously, resultsin cleavage of the amine group in the four position, resulting in theloss of its typical yellow coloration. The appearance of the localizedbiotinylation product and the lack of yellow color in the arterial wallof the experimental group (naphthalimide-modified heparin and light)provided additional evidence of successful bonding within tissue. Thelack of yellow indicated successful photochemical modification of thenaphthalimide, as breaking of the bond in the 4-amino position resultsin loss of the color and frees both the naphthalimide ring and thetethered heparin to bind to the tissue.

The results indicate that successful photochemical tethering of aclinically useful pharmacological agent, heparin, to arterial walltissue with a 4-amino-1,8-naphthalimide was achieved.

Example 17 Synthesis of Sunscreen-Modified Chitosan

To prepare phenylbenzimidazole sulfonyl chloride, 0.5 g ofphenylbenzimidazole sulfonic acid (274 g/mole) (1.8 mmoles) was added to50 ml of dry dioxane and stirred under reflux until dissolved. 0.21 g ofthionyl chloride (3.6 mmoles) was added to the reaction and the mixturerefluxed for 30 minutes. After 30 minutes the reflux condenser wasreplaced by a still head and the solvent was removed by distillation toa final volume of 5 ml of dioxane still in the flask.

The phenylbenzimidazole sulfonyl chloride was then added dropwise to 1gram of chitosan that had been dissolved in 20 ml of 10% acetic acid.This mixture was stirred for one hour, after which the pH was raised to8 by the dropwise addition of 1 M KOH with stirring. The mixture wasallow to react for three hours after which the modified chitosan wascollected by centrifugation, redissolved in dilute acetic acid, anddialyzed against PBS.

Example 18 Tethering of Sunscreen-Modified Biomolecule

To test the adherence of the sunscreen to the skin and the penetrationof the sunscreen into the skin, the skin of 6 female, Sprague Dawleyhairless rats were harvested. The naphthalimide (50 mg of Bradsyl in 1ml of acetone) was added at the same time as the phenylbenzimidazolesulfonyl chloride of Example 17 producing a sunscreen and naphthalimidemodified chitosan. The sunscreen prepared above was applied to 2×2 cmskin samples, allowed to dry and then washed to simulate swimming. A 40minute dry and 20 minute wash constituted one cycle. Different samplesunderwent up to six dry/wash cycles. The glove tips used to apply thesunscreen, the water washes, and an extraction of the sunscreen off skinwith dilute lactic acid were analyzed using the fluorescence of thetracer molecule to determine the amount of sunscreen that remainedadhered to the skin.

The penetration study also utilized the fluorescent tracer moleculeattached to the sunscreen. After various dry/wash cycles, the sampleswere preserved and processed for fluorescent microscopy analysis. Thefluorescent tracer demonstrated the degree of penetration of thesunscreen into the skin. The intensity of the fluorescent tracermolecule was also evaluated to determine how much sunscreen was adheredto the skin.

As shown in FIG. 24, the results of the study conclude that after aseries of six dry/wash cycles approximately 60% of the sunscreen remainstethered to the skin. The majority of the remaining 40% of the sunscreenwas lost on the glove tip during application or in the first water wash.The fluorescence microscopy verified that the sunscreen was onlyadhering to the epidermal surface and not penetrating into the skin.

REFERENCES CITED

The following U.S. Patent documents and publications are herebyincorporated by reference.

U.S. Pat. No. Issued to: 5,235,045 Lewis, et al. 5,565,551 Lewis, et al.5,766,600 Lewis, et al. 5,917,045 Lewis, et al. 6,410,505 Lewis, et al.

OTHER PUBLICATIONS

-   LeVeen, R., Wolf, G., Villanueva, T. New rabbit atherosclerosis    model for the investigation of transluminal angioplasty. Invest    Radiol, vol. 17, pp. 470-75, 1982.

1-16. (canceled)
 17. A method for stabilizing the wall of an arteryhaving arterial wall proteins, endogenous plaque, and a site of expandedlumen diameter after angioplasty, comprising: infusing an effectiveamount of a 4-amino-1,8-naphthalimide compound or its derivative intothe artery at the site of expanded lumen diameter; and activating the4-amino-1,8-naphthalimide compound or its derivative with a sufficientamount of an activating agent to cross-link the arterial wall proteinsand endogenous plaque and stabilize the wall of the artery.
 18. A methodfor local delivery of a pharmacological agent to a targeted tissue area,comprising: tethering the pharmacological agent to a4-amino-1,8-naphthalimide compound or its derivative to give a preparedspecies having a pharmacological agent moiety, a tether linkage, and anaphthalimide moiety; infusing the prepared species to the targetedtissue area, to give a delivered species; and activating the deliveredspecies with a sufficient amount of an activating agent to displace thenaphthalimide moiety and link the tether linkage to the tissue area. 19.The method of claim 18, wherein endogenous cleavage of the tetherlinkage releases the pharmacological agent moiety for cellularinteraction.
 20. A method for reducing the incidence of restenosis andstabilizing the wall of an artery having arterial wall proteins,endogenous plaque, and a site of expanded lumen diameter afterangioplasty, comprising: tethering an anti-restenotic agent to a4-amino-1,8-naphthalimide compound or its derivative to give a preparedspecies having an anti-restenotic agent moiety, a tether linkage, and anaphthalimide moiety; infusing the prepared species into the artery atthe site of expanded lumen diameter, to give a delivered species; andactivating the delivered species with a sufficient amount of anactivating agent to cross-link the arterial wall proteins and endogenousplaque, stabilize the wall of the artery, displace the naphthalimidemoiety, and link the tether linkage to the tissue area,
 21. The methodof claim 20, wherein endogenous cleavage of the tether linkage willrelease the anti-restenotic agent moiety for cellular interaction. 22.The method of claim 20, wherein the anti-restenotic agent is heparin,taxol, or sirolimus. 23-28. (canceled)
 29. The method of claim 17,wherein the 4-amino-1,8-naphthalimide compound has a structure selectedfrom the group consisting of:

wherein R, R′, and Q are straight-chain or branched chain alkyl groupshaving from 2 to 200 carbons.
 30. The method of claim 29, wherein thestraight-chain or branched chain alkyl groups further comprise one ormore ether, amide, or amine groups.
 31. The method of claim 18, whereinthe 4-amino-1,8-naphthalimide compound has a structure selected from thegroup consisting of:

wherein R, R′, and Q are straight-chain or branched chain alkyl groupshaving from 2 to 200 carbons.
 32. The method of claim 31, wherein thestraight-chain or branched chain alkyl groups further comprise one ormore ether, amide, or amine groups.
 33. The method of claim 20, whereinthe 4-amino-1,8-naphthalimide compound has a structure selected from thegroup consisting of:

wherein R, R′, and Q are straight-chain or branched chain alkyl groupshaving from 2 to 200 carbons.
 34. The method of claim 33, wherein thestraight-chain or branched chain alkyl groups further comprise one ormore ether, amide, or amine groups.